METHOD OF NATURAL EXPERIMENT

Fig. 40. Isophotophyll ofAllionia linearis, showing diphotic ecads: 1, light 1; 2, light .012; 3, light .003. × 130.

Fig. 41. Isophotophyll ofHelianthus pumilus, showing isophotic ecad: 1, sun leaf; 2, shade leaf (light .012). × 130.

179. Modification of the epidermis.The development of epidermal chloroplasts in diffuse light is the only change which is due to the direct effect of light. This does not often occur in the shade ecads of sun species, but chloroplasts are regularly present in the epidermis of woodland ferns and of submerged plants. The slight development of hairs in sciophilous plants is an advantage, but it must be referred to the factors that determine water loss. The significance of epidermal papillae in increasing the absorption of light by shade plants has already been discussed. The questions as to what factor has called forth these papillae and what purpose they serve must still be regarded as unsettled. The increased size of the epidermal cells, which is a fairly constant feature of shade ecads, seems to be for the purpose of increasing translocation and transpiration, and to bear no relation to light. The extreme development of the cells of the epidermis inStreptopusandLimnorchis, which grow at the edge of mountain brooks, has been plausibly explained by E. S. Clements as a contrivance to increase water loss. The presence of a waxy coating, such as that found upon the leaves ofImpatiens aureaandI. pallida, is clearly to prevent the wetting of the leaf and the consequent stoppage of the stomata. In regard to the latter, different observers have noted that the number of the stomata is greater in sun than in shade leaves. This holds generally for sun and shade species, but it is most clearly indicated by different ecads of the same species. InScutellaria brittonii, the sun form possesses 100 stomata per square millimeter, but in the shade these are reduced to 40 per square millimeter; the sun leaf ofAllionia linearishas 180 stomata to the square millimeter, the shade leaf 90. In the stable leaf ofErigeron speciosus, however, the number of stomata is the same, 180 per square millimeter, for sunlight and for diffuse light. The presence of the larger number of stomata in the plant exposed to greater loss, which at first thought seems startling, is readily explained by the more intense photosynthetic activity in the sun. Since the absorption of gases is the primary function of the stomata, and transpiration merely secondary, it is evident that sun plants must have more stomata than shade plants. This is further explained by the fact that the small air passages of sun leaves necessitate frequent inlets, which are less necessary in shade leaves with their larger air spaces. Inshade plants, moreover, the decrease in the number is compensated in some measure by the ability of the epidermal cells to absorb gases directly from the air.

Fig. 42. Diphotophylls ofQuercus novimexicana: 1, sun leaf; 2, shade leaf of the same tree (light .06). × 130.

180. The differentiation of the chlorenchym.The division of the chlorenchym into two tissues, sponge and palisade, is the normal consequence of the unequal illumination of the leaf surfaces. Exceptions to this rule occur only in certain monocotyledons, in which the leaf tissue consists of sponge-like cells throughout, and in those stable species that retain more or less palisade in spite of their change to diffuse light. The difference in the illumination of the two surfaces is determined by the position of the leaf. Leaves that are erect or nearly so usually have both sides about equally illuminated, and they may be termed isophotic. Leaves that stand more or less at right angles to the stem receive much more light upon the upper surface than upon the lower, and may accordingly be termed diphotic. Certain dorsiventral leaves, however, absorb practically as much light on the lower side as upon the upper. This is true of sun leaves with a dense hairy covering, which screens out the greater part of the light incident upon the upper surface. It occurs also in xerophytes which grow in light-colored sands and gravels that serve to reflect the sun’s rays upon the lower surface. In deep shade, moreover, there is no essential difference in the intensity of the light received by the two surfaces, and shade leaves are often isophotic in consequence. From these examples it is evident that isophotic and diphotic leaves occur in both sun and shade, and that the intensity of the light is secondary to direction, in so far as the modification of the leaf is concerned.

The essential connection of sponge tissue with diffuse light is conclusively shown by the behavior of shade ecads, but further evidence of great value is furnished by diphotic leaves, and those with hairy coverings. The sponge tissue, which in the shade leaf is due to the diffuse light of the habitat, is produced in the hairy leaf as a consequence of the absorption and diffraction of the light by the covering. In ordinary diphotic leaves, theabsorption of light in the palisade reduces the intensity to such a degree that the cells of the lower half of the leaf are in diffuse light, and are in consequence modified to form sponge tissue. The sponge tissue of the diphotic leaf is just as clearly an adaptation to diffuse light as it is in those plants where the whole chlorenchym is in the shade of other plants or of a covering of hairs. As is indicated later, all these relations permit of ready confirmation by experiment, either by changing the position of the leaf or by modifying the intensity or direction of the light.

Fig. 43. A plastic species,Mertensia polyphylla, showing the effect of water upon the sponge: 1, chresard 25%; 2, chresard 12%. × 130.

The preceding discussion makes it fairly clear that sponge tissue is developed primarily to increase the light-absorbing surface. Because of its direct connection with photosynthesis, the sponge tissue is the especial organ of aeration, also, and since it shows a high development of air spaces for this purpose, it is inevitably concerned in transpiration. It seems to be partly a coincidence, however, that the sponge is found next to the lower surface upon which the stomata are most numerous. This is indicated by artificial ecads ofRanunculus sceleratus, in which sponge tissue is unusually developed, although the stomata are much more numerous upon the upper surface. Palisade tissue is apparently developed primarily as a protection against water loss, particularly that due to the absorption of light by the chloroplast. The small size of the intercellular passages between palisade cells likewise aids in decreasing transpiration. The fact that leaves with much palisade tissue transpire twice as much as shade leaves is hardly an objection to this view, as Hesselmann (l. c., 442) would think. It is readily explained by the intense photosynthesis of sun plants, which makes necessary an increase, usually a doubling, in the number of stomata, in consequence of which the transpiration is increased.

Fig. 44. A stable species,Erigeron speciosus: 1, sun leaf; 2, shade leaf (light .03). × 130.

Fig. 45. Spongophyll ofGyrostachys stricta(light 1). × 130.

181. Types of leaves.Isophotic leaves are equally illuminated and possess more or less uniform chlorenchym. Diphotic leaves are unequally illuminated, and exhibit a differentiation into palisade and sponge tissues. They may be distinguished as isophotophylls and diphotophylls respectively.[18]Isophotic leaves fall into three types based upon the intensity of the light. The staurophyll, or palisade leaf, is a sun type in which the equal illumination is due to the upright position or to the reflection from a light soil, and in which the chlorenchym consists wholly of rows of palisade cells. The diplophyll is a special form of this type in which the intense light does not penetrate to the middle of the leaf, thus resulting in a central sponge tissue, or water-storage tissue. The spongophyll, or sponge leaf, is regularly a shade type; the chlorenchym consists of sponge cells alone. For the present at least it is also necessary to refer to this group those monocotyledons which grow in the sun but contain no palisade tissue. Diphotic leaves always contain both palisade and sponge, though the ratio between them varies considerably. Diphotophylls are characteristic of sunny mesophytic habitats. They are frequent in xerophytic habitats as well as in woodlands where the light is not too diffuse. In the case of stable species, this type of structure sometimes persists in the diffuse light of coniferous forests. Floating leaves, in which the light is almost completely cut off from the lower surface, are also members of this group. Submerged leaves, on the other hand, are spongophylls.

182. Heliophytes and sciophytes.The great majority of sun plants possess diphotophylls. This type is represented byPedicularis procera(fig. 32). Plants with isophotophylls are found chiefly in xerophytic places, though erect leaves of this type occur in most sunny habitats. The staurophyll, in which the protection is due to the extreme development of palisade tissue, is illustrated byAllionia linearis(fig. 40) andBahia dissecta(fig. 33). The diplophyll, which is characterized by a central band of sponge tissue or storage cells, is found inMertensia linearis(fig. 34). The form of the spongophyll that is found in certain monocotyledons is shown byGyrostachys stricta(fig. 45). The spongophyll (fig. 38:3, 39:2) is frequent among plants of deep shade, but as the leaf sections ofAllionia(figs. 38, 40) andQuercus(fig. 42) show, the diphotophyll is the rule in shade ecads.

183. Scope.The primary task of experimental evolution is the detailed study, under measured conditions, of the origin of new forms in nature. As a department of botanical research that is as yet unformed, it has little concern with the host of hypotheses and theories which rest merely upon general observation and conjecture. A few of these constitute good working hypotheses or serve to indicate possible points of attack, but the vast majority are worthless impedimenta which should be thrown away at the start. It is the general practice to speak of evolution as founded upon a solid basis of incontestible facts, but a cursory examination of the evidence shows that it is drawn, almost without exception, from observation alone, and has in consequence suffered severely from interpretation. With the exception of De Vries’s work on mutation, sustained and accurate investigation of the evolution of plants has been lacking. As a result, botanical research has been built high upon an insecure foundation, nearly every stone of which must be carefully tested before it can be left permanently in place. In a field so vast and important as evolution, experiment should far outrun induction, and deduction should enter only when it can show the way to a working hypothesis of real merit. The great value of De Vries’s study of mutation as an example of the proper experimental study of evolution has been seriously reduced by the fact that the “mutation theory” has carried induction far beyond the warrant afforded by experiment. The investigator who plans to make a serious study by experiment of the origin of new plant forms should rest secure in the conviction that the most rapid and certain progress can be made only by the accumulation of a large number of unimpeachable facts, obtained by the most exact methods of experimental study.

The general application of field experiment to evolution will render the current methods of recognizing species quite useless. It will become imperative to establish an experimental test for forms and species, and toapply this test critically to every “new species.” Descriptive botany, as practiced at present, will fall into disuse, as scientific standards come to prevail, and in its place will appear a real science of taxonomy. In the latter the criteria upon which species are based will be obtained solely by experiment.

184. Fundamental lines of inquiry.There are two primary and sharply defined fields of research in experimental evolution, namely, adaptation in consequence of variation (and mutation), and hybridization. The latter constitutes a particular field of inquiry, which is not intimately connected with the problems of evolution in nature. In the study of specific adaptation, two questions of profound importance appear. One deals with the effects of ancestral fixity or plasticity in determining the amount of modification produced by the habitat. These are fundamental problems, and a solution of them can not be hoped for until exact and trustworthy data have been provided by numerous experimental researches. It thus becomes clear that the principal, if not the sole task of experimental evolution for years to come is the diligent prosecution of accurate and prolonged experiment in the modification of plant forms. It seems inevitable that this will be carried on along the lines that have already been indicated. Plants will be grown in habitats of measured value, or in different intensities of the same factor. The relation between stimulus and adjustment will form the basis of careful quantitative study, and the final expression of this relation in structural modifications will find an exact record in drawings, photographs, exsiccati, and biometrical measures. The making of an accurate and complete record of the whole course of each experiment of this sort is an obligation that rests upon every investigator. Studies in experimental evolution will prove time-consuming beyond all other lines of botanical research, and the work of one generation should appear in a record so perfect that it can be used without doubt or hesitation as a basis for the studies of the succeeding generation.

185. Ancestral form and structure.The significance of the fact that some species have been found to remain unaltered structurally under changes of habitats that produced striking modifications in others has already been commented upon. It is hardly necessary to indicate the important bearing which this has upon evolution. The very ability of a plant to undergo modification, and hence to give rise to new forms, depends upon the degree of fixity of the characters which it has inherited. Stable plants are less susceptible of evolution than plastic ones. The latter adapt themselves to new habitats with ease, and in each produce a new form, which may serveas the starting point of a phylum. There is at present no clue whatever as to what calls forth this essential difference in behavior. This is not surprising in view of the fact that there have been no comparative experimental studies of stable and plastic species. Until these have been made, it is impossible to do more than to formulate a working hypothesis as to the effect of stability, and an explanation of the forces which cause or control it is altogether out of the question.

186. Variation and mutation.New forms of plants are known to arise by three methods, viz., variation, mutation, adaptation. The evidence in support of these is almost wholly observational, and consequently more or less inexact, but for each there exist a few accurate experiments which are conclusive. Origin by variation and subsequent selection is the essence of the Darwinian theory of the origin of species. According to this the appearance of a new form is due to the accumulation, and selection, through a long period, of minute differences which prove advantageous to the plant in its competition with others in nature, or are desirable under cultivation. Slight variations appear indiscriminately in every species. Their cause is not known, but since they are found even in the most uniform habitats, it is impossible to find any direct connection between them and the physical factors. In the case of origin by mutation, the new form appears suddenly, with definite characteristics fully developed. Selection, in the usual sense of the term, does not enter into mutation at all, though the persistence of the new form is still to be determined by competition. Mutations are known at present for only a few species, and their actual appearance has been studied in a very few cases. Like variations, they are indiscriminate in character. The chief difference between them is apparently one of degree. Indeed, mutation lends itself readily to the hypothesis that it is simply the sudden appearance of latent variations which have accumulated within the plant. De Vries regards constancy as an essential feature of mutation, but the evidence from the mutants ofOnagrais not convincing. Indeed, while there can be no question of the occurrence of mutation in plants, a fact known for many years, the facts so far brought forward in support of the “mutation theory” fall far short of proving “the lack of significance of individual variability, and the high value of mutability for the origin of species.”[19]Mutations do not show any direct connection with the habitat, but their sudden appearance suggests that they may be latent or delayed responses to the ordinary stimuli. Origin by adaptation is the immediate consequence of the stimuli exerted by the physical factors of ahabitat. This fact distinguishes it from origin by variation, or by mutation. The new form may appear suddenly, often in a single generation, or gradually, but in either case it is the result of adaptation that is necessarily advantageous, because it is the result of adjustment to controlling physical factors. Origin by adaptation is perhaps only a special kind of origin by variation, but this might be said with equal truth of mutation. New forms resulting from adaptation are like those produced from mutation, in that they appear suddenly as a rule and without the agency of selection. They are essentially different, inasmuch as their cause may be found at once in the habitat, and since a reversal of stimuli produces, in many cases at least, a reversion in form and structure to the ancestral type.

A valid distinction between forms or species upon the basis of constancy is impracticable at the present time. It is doubtful that such a distinction can ever be made in anything like an absolute sense, since all degrees of fluctuation may be observed between constancy and inconstancy. In all events, it is gratuitous to make constancy the essential criterion in the present state of our knowledge. So little is certainly known of it that it is equally unscientific to affirm or to deny its value, and even a tentative statement can not be ventured until a vast amount of evidence has been obtained from experiment. Accordingly, there is absolutely no warrant, other than tradition, for limiting the term species to a constant group. In the evolutionary sense, a species is the aggregate ancestral group and the new forms which have sprung from it by variation, mutation, or adaptation. It should not be regarded as an isolated unit for purposes of descriptive botany; indeed, its use in this connection is purely secondary. It is properly the unit to be used in indicating the primary relationships which are the result of evolution.

On the basis of their actual behavior in the production of new forms, species may be distinguished as variable, mutable, or adaptable. The new form which results from variation is avariant; the product of mutation is amutant, and that of adaptation, anecad. The following examples serve to illustrate these distinctions.Machaeranthera canescens, judging from the numerous minute intergrades between its many forms, is a variable species, i. e., one in which forms are arising by the gradual selection of small variations. It apparently comprises a large number of variants,M. canescens aspera,superba,ramosa,viscosa, etc.Onagra lamarckianais a mutable species: it comprises many mutants, e. g.,Onagra lamarckiana gigas,O. l. nanella,O. l. lata, etc.Galium borealeis an adaptable species: it possesses one distinct ecad,Galium boreale hylocolum, which is the shade form of the species.

187. Methods.The best of all experiments in evolution are those that are constantly being made in nature. Such experiments are readily discovered and studied in the case of origin by adaptation; variants present much greater difficulties, while mutants are very rare under natural conditions. The method which makes use of these experiments may be termed themethod of natural experiment. The number of ecads which appear naturally in vegetation is limited, however, and it is consequently very desirable to produce them artificially, by themethod of habitat culture. This method, while involving more labor than the preceding, yields results that are equally conclusive, and permits the study of practically every species. Themethod of control culture, which is carried on in the planthouse, naturally does not possess the fundamental value of the field methods. It is an invaluable aid to the latter, however, since it permits the physical factors to be readily modified and controlled. All these methods are based on the indispensable use of instruments for the measurement of physical factors.

188. Selection of species.Species that are producing variants or ecads are found everywhere in nature; those which give rise to mutants seem, however, to be extremely rare. Consequently, mutants can not be counted upon for experimental work, and their study scarcely needs to be considered. When a mutant is discovered by some fortunate chance, the mutable species from which it has sprung, and related species as well, should be subjected to the most critical surveillance, in the hope that new mutants will occur or the original one reappear. On account of the suddenness with which they appear, mutants do not lend themselves readily to natural experiment, and after they have once been discovered, inquiry into the causes and course of mutation is practicable only by means of habitat and control cultures. Among variable species, those are most promising that show a wide range of variation and are found in abundance over extensive areas. A species which occurs in widely separated, or more or less isolated areas, furnishes especially favorable material for investigation, since distance or physical barriers partly eliminate the leveling due to constant cross-fertilization. The individuals or groups which show appreciable departure from the type are marked and observed critically from year to year. The direction of the variation and the rapidity with which small changes are accumulated can best be determined by biometrical methods. Representative individuals of the species and each of its variants should likewise be selected from year to year. After being photographed, these are preserved as exsiccati, and with the photographs constitute a completegraphic record of the course of variation. When the latter is made evident in structural feature also, histological slides are an invaluable part of the record.

Polydemic species are by far the best and most frequent of all natural experiments. In addition to plants that are strictly polydemic, i. e., grow in two or more distinct habitats, there are a large number which occur in physically different parts of the same habitat. The recognition of polydemics is the simplest of tasks. As a rule, it requires merely a careful examination of contiguous formations in order to ascertain the species common to two or more of them. The latter are naturally most abundant along the ecotones between the habitats, and, as a result, transition areas and mixed formations are almost inexhaustible sources of ecads. Many adaptable species are found throughout several formations, however, and such are experiments of the greatest possible value. Not infrequently species of the manuals are seen to be ecads, in spite of their systematic treatment, and to constitute natural experiments that can be readily followed. Finally, it must be kept in mind that some polydemics are stable, and do not give rise to ecads by structural adaptation. They not only constitute extremely interesting experiments in themselves, but they should also be very carefully followed year by year, since it seems probable that the responses are merely latent, and that they will appear suddenly in the form of mutants. In natural experiments it is sometimes difficult to distinguish which form is the ecad and which the original form of the species. As a rule, however, this point can be determined by the relative abundance and the distribution, but in cases of serious doubt, it is necessary to appeal to experimental cultures.

Although habitats differ more or less with respect to all their factors, the study of polydemics needs to take into account only the direct factors, water-content, humidity, and light. Humidity as a highly variable factor plays a secondary part, and in consequence the search for ecads may be entirely confined to those habitats that show efficient differences in the amount of water-content or of light. Temperature, wind, etc., do not produce ecads, and may be ignored, except in so far as they affect the direct factors. Complexes of factors, such as altitude, slope, and exposure, are likewise effective only through the action of the component simple factors upon water and light. The influence of biotic factors is so remote as to be negligible, especially in view of the fact that ecads are necessarily favorable adaptations, and are in consequence little subject to selective agencies. The essential test of a habitat is the production of a distinguishable ecad, but a knowledge of the water-content and light values of the habitats under examination is a material aid, since a minute search of each formation is necessary to reveal all the ecads. It is evident that habitats orareas that do not show efficient differences of water or light will contain no ecads of their common species, and also that extreme differences in the amount of either of these two factors will preclude origin by adaptation to a large degree, on account of the need for profound readjustment. The general rule followed by most polydemics is that sun species will give rise to shade forms, and vice versa, and that xerophytes will produce forms of hydrophytic tendency, or the converse, when the areas concerned are not too remote, and the water or light differences are efficient, but not inhibitive. Some species are capable of developing naturally two series of ecads, one in response to light, the other to water-content, but they, unfortunately, have been found to be rare. Greatly diversified regions, such as the Rocky mountains, in which alternation is a peculiarly striking feature of the vegetation, are especially favorable to the production of ecads, and hence for the study of natural experiments in origin by adaptation.

189. Determination of factors.For the critical investigation of the origin of new forms, an exact knowledge of the factors of the habitat, both physical and biotic, is imperative. In the case of variable species, these factors determine what variations are of advantage, and thereby the direction in which the species can develop. They are the agents of selection. With mutants, the factors of the habitat are apparently neither causative nor selective, though it seems probable that further study of mutants will show an essential connection between mutant and factor. In any event, the persistence of a mutant in nature, and its corresponding ability to initiate new lines of development, is as much dependent upon the selection exerted by physical and biotic factors as is the origin of variants. Physical factors are causative agents in the production of ecads, as has been shown at length elsewhere. The form and structure of the ecad are the ultimate responses to the stimuli of light or water-content, and the quantitative determination of the latter is accordingly of the most fundamental importance. The measurement of factors has been treated so fully in the preceding chapters that it is only necessary to point out that the thorough investigation of habitats by instruments is as indispensable for the study of experimental evolution as for that of the development and structure of the formation. Furthermore, it is evident that a knowledge of physical factors is as imperative for habitat and control cultures as for the method of natural experiment. In the latter, however, the biotic factors demand unusual attention, since pollination, isolation, etc., are often decisive factors in origin by variation and in the persistence of mutants.

Measurements of adjustment, i. e., functional response to the direct factor concerned, are extremely valuable, but not altogether indispensableto research in experimental evolution. This is due to the fact that a knowledge of adjustment is important in tracing the origin of new forms only when adjustment is followed by adaptation, and in all such cases the ratio between the two processes seems to be more or less constant. In the present rudimentary development of the subject, however, it is very desirable to make use of all methods of measuring functional responses to water and light that are practicable in the field. Certain methods that are difficult of application in nature may be used to advantage in control cultures, and the results thus secured can be used to interpret those obtained from natural experiments and field cultures.

190. Method of record.As suggested elsewhere, there are four important kinds of records, which should be made for natural experiments, and likewise for habitat and control cultures. These are exsiccati, photographs, biometrical formulae and curves, and histological sections. These serve not merely as records of what has taken place, but they also make it possible to trace the course of evolution through a long period with an accuracy otherwise impossible, and even to foreshadow the changes which will occur in the future. The possibility of doing this depends primarily upon the completeness of the record, and for this reason the four methods indicated should be used conjointly. In the case of ecads and mutants, exsiccati, photographs, and sections are the most valuable, and in the majority of cases are sufficient, since both ecads and mutants bear a more distinctive impress than variants do. On the other hand, since variations are more minute, the determination of the mean and extreme of variation by biometrical methods is almost a prerequisite to the use of the other three methods, which must necessarily be applied to representative individuals.

Exsiccati and photographs are made in the usual way for plants, but it is an advantage to photograph each ancestral form alongside of its proper ecads, mutants, or variants, in addition to making detail pictures of each form and of the organs which show modification. In the collection of material for histological sections, which deal primarily with the leaf or with stems in the case of plants with reduced leaves, a few simple precautions have been found necessary. Whenever possible, material should be killed where it is collected, since in this way the chloroplasts are fixed in their normal position. In case leaves that can not be replaced easily have become wilted, an immersion of 5–6 hours in water will make it possible to kill them without shrinkage. In selecting leaves, great pains must be taken to collect only mature leaves. When the plants have a basal rosette, or distinct radical leaves, mature leaves are taken from both stem and base. In all cases where the two surfaces of the leaf can not be readily distinguished, the upper one is clearly marked.

METHOD OF HABITAT CULTURES

191. Scope and advantages.By means of experiments actually made in the field, practically every species that is capable of modification can be made to produce new forms, the origin of which can be traced in the manner already indicated. Field experiments of this sort are especially favorable to the production of ecads from adaptable species. No attempt has yet been made to apply it to mutable or variable species, but its ultimate application to these does not seem at all impossible. The chief advantage of the method of habitat cultures is seen in the great range of choice in selecting the plant for experiment, and the habitat or area in which the experiment is carried out. A polydemic species which already has one or more ecads can be extended to a number of different habitats of known value, and a complete series of ecads obtained, based either upon water-content, or light, or upon both. On the other hand, an endemic species, or one brought from a remote flora, can be placed in as many habitats as desired, and the appearance of ecads followed in each. Frequently, results of much value are obtained in a diversified habitat by growing its most plastic species in those areas which show the greatest differences in water-content or light intensity. Habitat cultures give results which are practically as perfect as those obtained from natural experiments, since the course of adaptation in no wise depends upon whether the agent by which the seed or propagule is carried into the new habitat is natural or artificial. Cultures of this kind further possess the distinct advantage of permitting more or less modification of the physical factors themselves. However, when it is desirable to have the factors under as complete control as possible, it is necessary to use the method of control cultures in the planthouse.

192. Methods.All field experiments in evolution are based upon a change of habitat. The latter is accomplished by the modification of the habitat itself, or by the transfer of the species to one or more different habitats, or to different areas of the same habitat. In both cases the choice of habitats is made upon the basis of efficient differences of water-content or light. Saline situations do not constitute an exception, since the chresard is really the effective stimulus. Cultures at different altitudes, which afford striking results, appear to concern several factors, but in the final analysis, water-content and humidity are alone found to be really formative. Cultures may furthermore be distinguished as simple or reciprocal. Simple cultures are those in which a species is transferred to one or more habitats, or in which a habitat is modified in one or more ways. Reciprocal cultures are possible only with polydemic species, or with endemics after ecads havebeen produced by experiment. Modification or transfer is made in the usual way, but reciprocally, i. e., the original form is transferred to the habitat of the ecad, and the latter to the habitat of the former; or the shade in which some individuals of the ecad are growing may be destroyed, and at the same time individuals of the type may be shaded. Both transfer and modification may be applied to the same species, but since the same measured change of factor can be obtained in either way, the use of both is undesirable, with the exception of the rare cases where they serve as checks upon each other. The transfer of a seed or plant is so much simpler and more convenient that this method is the one regularly used. It sometimes happens, however, that a change of water-content or light intensity is readily and conveniently made, and is desirable for other reasons.

It is evident that both transfer and modification require that the factor records of the various habitats or areas be as full as possible, at least so far as water-content, humidity, and light are concerned. In the case of the areas that are to be modified, these factors are determined before the change is made. Afterward they are read from time to time during the growing season, and are also checked by readings made near at hand in the unmodified formation. The readings made in the beginning should correspond closely to the check readings, but in case of disagreement the latter are to be taken as conclusive.

193. Transfer.After the species to be used for experiment has been chosen, the various habitats or areas selected, and the direct factors measured by instruments, the actual transfer of the individuals is made by means of seeds, preferably in autumn, though the results are practically the same if seeds are kept over the winter and planted at the opening of spring. The natural method is to scatter the seeds in the place selected, as though they had been carried by the usual agents of migration. The mortality is usually great in such case, however, and the chances of success are increased by actually planting the seeds. This is the method which has been used in making cultures of species of the European Alps on the summit of Mount Garfield in the Rocky mountains. The number of seeds used is recorded in order to obtain some estimate of germination and competition. While the use of the seed or disseminule possesses the great advantage of making the experiment essentially a natural one, the transfer of rosettes, seedlings, or young plants makes the results more certain, and consequently saves time, even though the actual transfer is somewhat more difficult. It is hardly necessary to point out that the removal of the plant should be made with the greatest care. The best success is obtained by making the transfer on cloudy or rainy days, and when shade plants are to be placed in sunny situations, they should be transplanted late in the afternoon. When the task of carryingthem is not too great, it is a distinct advantage to move a number of individuals in the same block of earth. The transfer of mature plants is inadvisable, except for those perennials which can not readily be secured in an early stage. This naturally does not apply to woody plants, evergreen herbs, mosses and lichens; the last two may be transferred at any time with satisfactory results. Each culture is carefully marked with stakes, and definitely located by means of landmarks.

Fig. 46. Series for producing hydrophytic forms under control: 1, amphibious; 2, floating; 3, competition; 4, submerged.

Reciprocal transfers may be made by means of seed or plant. Since the experiment is a complex one, all the care possible should be taken to make sure that the plants become established in the reciprocal situations, and consequently, it is often advisable to transfer both seeds and plants. Reciprocal transfer is of paramount value in solving the problem which bog plants present. A slight modification of the method makes it possible to obtain experimental evidence of the polyphyletic origin of species in consequence of adaptation. In an experiment mentioned elsewhere, the transfer ofKuhnistera purpureato the area occupied byK. candida, and vice versa, is designed to show whether one has been derived from the other. If the two species are moved into an area which contains more water than that usually occupied byK. purpurea, and less water than is found whereK. candidahabitually grows, the resulting modifications will throw much light upon theorigin of polyphyletic species. In this connection, it hardly needs to be pointed out that this simple transfer of a species to several separated areas of a new habitat may often furnish complete proof that a new form may arise at different times, and at different places.

Fig. 47. Control ecad ofRanunculus sceleratus, holard 10% (50 cc.).

194. Modification of the habitat.Efficient changes in the habitat are brought about by increasing or decreasing the water-content, or by varying the light intensity between sunshine and the diffuse light of deep forests. Humidity can not well be regulated except in so far as it is connected with water-content. Since its effects merge with those of the latter, its modification is unnecessary. An increase in water-content is readily brought about by irrigation. A stream may be dammed and its water allowed to spread over the area to be studied, or the water may be carried to the proper place by deflecting the stream or by digging a canal. The construction of earth reservoirs makes it possible to obtain almost any per cent of soil water by varying the size of the reservoir or the height of the wall or bank. Near a base station, such as Minnehaha, where there is a simple system of water-works, the experimental area may be watered whenever desirable by means of a hose. Water-content may be readily decreased by drainage, or by the deflection of a stream. When such means are not available, as in the case of extensive marshes, hummocks may be used or constructed, and the soil blocks containing plants placed upon them. By the use of sand or gravel, the water-content of mesophytic areas can be reduced in a similar manner, or by surrounding the plantin situwith either of these soils which hold little water. In meadows, especially, the addition of a large quantity of alkaline salts decreases the amount of available water, while the holard may be reduced by denuding the soil about the plants concerned.

Fig. 48. Control ecad ofRanunculus sceleratus, holard 40% (200 cc.).

In sunny habitats, the light intensity is most easily reduced by means of cloth awnings, which can be put in place conveniently. It is not a difficult matter to produce effective shade by using shrubs or small trees for this purpose. This plan is especially advantageous in habitats too remote to make frequent visits feasible. When a shrub or tree is used, the experiment necessarily requires a longer time, though this disadvantage is partly compensated by the fact that the shelter requires practically no attention after the shrub is once established. Forest plantations furnish excellent examples of this kind of experiment. On the other hand, clearings afford the only examples of habitats modified in such manner as to increase the light. In nature, the diffuse light in which shade plants grow is due to the presence of tall plants, chiefly shrubs and trees, and an increase in the light intensity is possible only through the thinning-out or removal of the plant screen. This is a task of considerable magnitude in forests, but it can be readily accomplished in thickets and at the edges of woodlands. It is quite practicable to establish a series of awnings or clearings of various light values, but the labor required is hardly worth while when it is recalled that the method of transfer makes it possible to take advantage of the various intensities already found in nature.

195. Scope and procedure.Control experiments are necessarily carried on in the planthouse, since factors can be controlled in the field only with great difficulty. Their greatest value is in connection withexperiments that are being carried on in the habitat, but they also constitute an invaluable means of independent research, since it is not at all difficult to approximate the conditions of a habitat, especially with reference to water-content and light. The essential feature of the method is that the less important factors are equalized as far as possible, while the direct factors, water-content and light, are under the complete control of the investigator. By the equalization of humidity and temperature is meant experimentation in which all the plants of each experiment are subjected to the same amounts of these factors. It is a matter of no importance whatever whether the humidity and temperature are constant or variable. In the case of soil, which is not a variable, it naturally happens that the plants are placed once for all in the same soil mixture. Batteries consisting of thermograph and psychrograph have been kept in the different control houses, but although used at first to give some idea of the hourly and daily fluctuations of temperature and humidity, they have slight bearing upon the evolution of new forms under control. For use in connection with supplementary experiments in adjustment and adaptation, the batteries have proved to be indispensable. Control experiments are regularly made in series which are planned with reference to as many modifications as the efficient difference of the factor and the plasticity of the species concerned permit.

196. Water-content series.An account of the experiments which have been carried on for four generations withRanunculus sceleratuswill serve to show the application of culture methods to the origin of new forms in response to varying water-content. This species was chosen because it grows readily in the planthouse, is plastic, and, since it is naturally amphibious, permits of much modification in both directions. The smallest amount of water per day under which the seedlings would grow was found to be 25 cc. This was taken as one extreme for the series, and deep water in which the plant could be submerged as the other. An arbitrary series was tentatively made as follows: 25 cc., 50 cc., 100 c., 150 cc., 200 cc., mud, shallow water, and deep water. Further study justified these divisions, since the first six gave efficient differences in water-content, and the resulting forms all showed differences of structure as well as of growth and form. Seedlings of the same age, and as nearly alike as possible, were transplanted to large pots of which there were four for each of the first six; they were placed in half-barrels for mud and floating forms, and in a barrel for submerged forms. After a few days, when they had become well established, the plants in the pots were watered in the amounts indicated, as often as was necessary to keep the most xerophytic form alive; the soil forthe mud form was kept covered with a thin film of water; the leaves of the form in shallow water were kept floating on the surface, and those of the last form submerged just below the surface. The water in which the submerged form grew was aerated by means of a spigot near the bottom of the barrel. From time to time water-content determinations were made of the soil in the pots until it was definitely ascertained that the holard was practically constant. The nine new forms obtained by adaptation showed striking differences in vigor and growth, as may be seen from the figures. In all cases, these were accompanied by distinct and often striking differences in the number and position of the stomata, the amount of sponge and palisade tissues, and the development of air passages. Photographs were made of a typical plant of each form, and the different leaf structures were preserved in permanent mounts. The xerophytic and the submerged form were unable to produce flowers, and it was necessary to develop them anew in each generation. The other forms fruited abundantly, and the succeeding generations of each form were produced from plants which had grown the year before in the same conditions. In addition to the development of a series of new water-content forms, this experiment was begun in the hope of determining whether the modifications of a plastic species tend to become fixed if each new form is grown constantly under the same conditions. A period of four years is too short, however, to throw much light upon this problem.

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